BIOACTIVITY COMPOSITION OF Reevesia formosana

ABSTRACT

A series cardenolide derivatives including structure of formula I from the root of  Reevesia formosana  have provided. In formula (1) and (2), where R 3 , R 5 , R 10  and R 16  are as defined in the specification. The derivatives compounds showed potent cytotoxicity against MCF-7, NCI-H460, and HepG2 cancer cell lines.

The application claims the benefit of Taiwan Patent Application No.100112521, filed on Apr. 11, 2011, in the Intellectual Property Officeof Republic of China, the disclosure of which is incorporated byreference as if fully set forth herein.

FIELD OF THE INVENTION

The present invention relates to a compound, in particular, tocardenolide derivatives from the root of Reevesia formosana, whichshowed the cytotoxic effects on cancer cells.

BACKGROUND OF THE INVENTION

Reevesia formosana Sprague is an endemic deciduous tree grows insouthern Taiwan. There are about 25 species of Reevesia worldwide, withtwo species in central America, fourteen in mainland China, and one inTaiwan. The remainder are found mostly in Southeast Asia. In 2003, therewere five known compounds which are β-sitosterol, daucosterol, betulinicacid, lupeol, (+)-catechin isolated from R. longipetiolata.

Nowadays, in research of natural, a separating strategy is usually putemphasis on active components. A collected natural is extracted byalcohol or aqueous alcohol, and the extractives are separated intofractions by different ionizing solvent partitions. Then, the eachfraction is examined by every kind of biological activity test, and thefraction with active components is further examined by separating andextracting techniques to acquire physiological activities. Suchexamining model combined chemical components analyses and activityanalyses is called bioassay-guided fractionation.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, apharmaceutical composition is provided and includes an effective amountof a pharmaceutical compound being represented by formula 1:

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R⁵ group is one of a hydrogen radical and a hydroxyl group, the R¹⁰group is one selected from a group consisting of a hydrogen radical, ahydroxyl group, a methyl radical, a formaldehyde group and a formic acidradical, and the R¹⁶ group is one of a hydrogen radical and an aceticacid radical.

In accordance with a second aspect of the present invention, apharmaceutical composition is provided and includes an effective amountof a pharmaceutical compound being represented by formula 2:

According to the above-mentioned aspects of the present invention,collected roots of Reevesia formosana were dried and cut into slices.Then, the slices were extracted by methanol in the room temperature.After eliminating the solvent, the extractive was partitioned into anEtOAc fraction and a water fraction by EtOAc and H₂O, wherein the EtOAcfraction was subjected to a silicon column, and a concentration gradientof n-hexane-EtOAc was applied on elution to acquire 1˜12 fractions, andat last methanol was used to elute to form 13 fractions totally. TheEtOAc fraction and the water fraction were conducted active examinationsindividually, and the result showed that the extractives have ancytotoxic effect on tumor cells of MCF-7, NCI-H460 and HepG2. Table. 1shows that the fractions 9˜13 have stronger cytotoxic effects on thetumor cells of MCF-7, NCI-H460 and HepG2.

TABLE 1 The survival rates of EtOAc fractions 9~13 Cell line\fraction 910 11 12 13 MCF-7 150 (μg/ml)  9% 4%  8%  8%  8%  30 (μg/ml) 38% 5%  6% 6%  5% NCI- 150 (μg/ml)  8% 5% 91% 10% 12% H460  30 (μg/ml) 44% 9% 10%10%  9% HepG2 150 (μg/ml)  4% 2%  2%  2%  3%  30 (μg/ml)  2% 1%  1%  1% 1%

According to the above-mentioned aspects of the present invention,materials of the column were selected from silicon, Sephadex LH-20,RP-C₁₈ etc. the following elution and purification were conducted viasolvents of CH₂Cl₂, EtOAc, n-hexane, methanol, acetone, water and themixtures thereof. See Table. 2, wherein the fraction 10 which wassubjected to passage over a Sephadex LH-20 column, eluting with 100%methanol, to yield 13 fractions (fractions 10-1 to 10-13). The fraction10-3 was separated over a RP-C₁₈ column, eluting with a solvent mixed bythe methanol and the water with a ratio 3:2, and fractions 10-3-1 to10-3-12 were obtained.

The fraction 10-3-6 was subjected to the silicon column, eluting with amixing solvent of CH₂Cl₂-acetone (3:1), to furnish 7 fractions(fractions 10-3-6-1 to 10-3-6-7). The fraction to 10-3-10 was purifiedby the silicon column, eluting with a mixing solvent of CH₂Cl₂-acetone(3:1) to yield 5 fractions (fractions 10-3-10-1 to 10-3-10-5). Thefraction 11 was applied to a Sephadex LH-20 column, eluting with 100%methanol, to afford 9 fractions (fractions 11-1 to 11-9). The fraction11-2 was submitted to a RP-C₁₈ column, using a mixing solvent ofmethanol-water (1:1) for elution, to obtain 14 fractions (fractions11-2-1 to 11-2-14). The fraction 11-2-8 was subjected to passage over asilicon column, eluting with a mixing solvent of CH₂Cl₂-acetone (3:1),to yield 9 fractions (fractions 11-2-8-1 to 11-2-8-9).

Besides, compound 1 (460 mg) was separated from the fraction 10-3-8, anda crystal mixture (10 mg) of compounds 8 and 9 was acquired from thefraction 10-3-5 by eluted with the solvent of n-hexane-EtOAc. Afterfiltering out the crystal, the solution was subjected to the siliconcolumn eluting with the mixing solvent of CH₂Cl₂-acetone (5:1), toobtain 7 fractions (fractions 10-3-5-1 to 10-3-5-7). The fraction10-3-6-2 was purified by the RP-C₁₈ manufacturing TLC plate via themixing solvent of acetone-water (1:1), and then compound 2 (2 mg) wasacquired. The fractions 11-2-8-7 and 11-2-8-8 were purified by themanufacturing TLC plate via the mixing solvent ofn-hexane-CH₂Cl₂-acetone (1:1:2), and then compounds 3 (9 mg), 4 (14 mg),5 (2.8 mg) and 6 (11 mg) were acquired. The fraction 10-3-10-3 andfraction 10-3-10-4 contained compounds 7 (63 mg) and 6 (11 mg),respectively. The fraction 10-3-5-2 contained a mixture (1.7 mg) ofcompounds 12 and 13, and the fraction 10-3-5-5 contained a mixture (7mg) of compounds 10 and 11.

TABLE 2 A EtOAc solvable fraction was subjected to the silicone column.solvent n-hexane fraction 1 n-hexane/EtOAc fractions 2~9(90:10~85:15~80:20~70:30~50:50) EtOAc fractions 10~12 methanol fraction13 B fraction 10 was subjected to passage over a Sephadex LH-20 column.solvent methanol fractions 10-1 to 10-13 C fraction 10-3 was separatedover a RP-C₁₈ column. solvent methanol:water (3:2) fractions10-3-1~10-3-12 D Filter out the crystal mixture of compound 8 and 9, andthe solution was subjected to the silicone column. solventCH₂Cl₂-acetone (5:1) fractions 10-3-5-1~10-3-5-7 E fraction 10-3-6 wassubjected to the silicone column. solvent CH₂Cl₂-acetone (3:1) fractions10-3-6-1~10-3-6-7 F fraction 10-3-6-2 was purified by RP-18manufacturing TLC plate. solvent acetone-water (1:1) G fraction 10-3-10was purified by the silicone column. solvent CH₂Cl₂-acetone (3:1)fractions 10-3-10-1~10-3-10-5 H fraction 11 was applied to a SephadexLH-20 column. solvent methanol fractions 11-1 to 11-9 I fraction 11-2was submitted to a RP-C₁₈ column. solvent methanol:water (1:1) fractions11-2-1 to 11-2-14 J fraction 11-2-8 was subjected to passage over asilicone column. solvent CH₂Cl₂-acetone (3:1) fractions 11-2-8-1 to11-2-8-9

The separated and purified compounds were individually examined by aYanaco micromelting apparatus, by related equipments to test NMR of UV,KBr, ¹H and ¹³C and by single-crystal x-ray crystallography to confirmthe structures of compounds. As shown in FIG. 1 is a monocrystal x-raydiffractogram of compound 1.

The NMR spectra of ¹H and ¹³C of the compounds 10 and 11 were similar tospectra of the compounds 8 and 9, except that the aldehyde group [δ_(H)10.05 (1H, s, H-19); δ_(c) 207.9 (C-19)] of 8 and 9 was replaced by acarboxylic acid group [δ_(c) 176.0 (C-19)] in 10 and 11. The compounds12 and 13 were also duplicated the pattern of ¹H and ¹³C NMR signalsconcerning the 18,20-epoxide structures of 8 and 9.

Cardenolides are found in Apocynaceae, Asclepiadaceae, Asteraceae,Brassicaceae, Euphorbiaceae, Liliaceae, Moraceae, Ranunculaceae, andScrophulariaceae. Via the bioassay-guided-fractionation, the collectedroot of Reevesia formosana was demonstrated that its EtOAc and waterfractions have cytotoxic effects on human breast adenocarcinoma (MCF-7)non-small-cell lung cancer (NCI-H460) and liver hepatocellular cells(HepG2). The purified cardenolide derivatives can be divided into thecompounds as presented in formula 1 and 2 according to the physical andchemical properties (Tables. 4˜9), wherein the compounds 8 and 9 weremixed with ratio 6:5, and the compounds 10 and 11, and the compounds 12and 13 were both mixed with ratio 3:2. As shown in Table. 3, thecardenolide derivatives have cytotoxic effects on tumor cells of MCF-7,NCI-H460 and HepG2.

TABLE 3 IC₅₀ of 13 cardenolide derivatives IC₅₀ (nM) Compounds MCF-7NCI-H460 HepG2 reevesioside A (1) 63.2 ± 2.1 19.3 ± 0.5 367.6 ± 29.9reevesioside B (2) 3524.3 ± 72.0  431.1 ± 45.1 4755.3 ± 202.5reevesioside C (3) 2036.2 ± 172.8 207.9 ± 15.6 3738.4 ± 261.1reevesioside D (4) 36427.0 ± 2577.5 3904.4 ± 316.2 >500000 reevesiosideE (5) 11764.2 ± 2239.4 1769.6 ± 35.4  40491.5 ± 6425.4 reevesioside F(6) 72.0 ± 8.1 20.3 ± 0.4 836.2 ± 33.2 epi-reevesioside 34.1 ± 5.5 10.4± 0.5 995.5 ± 92.6 F (7) mixture of 2070.8 ± 52.6  485.6 ± 29.3 8903.9 ±790.7 reevesioside G (8) and epi- reevesioside G (9) mixture of 36194.2± 4128.6 3665.3 ± 182.5 >500000 reevesioside H (10) and epi-reevesioside H (11) mixture of 9352.9 ± 962.9 1090.8 ± 207.0 22739.3 ±3489.7 reevesioside I (12) and epi- reevesioside I (13) tylophorine*236.0 ± 11.0 233.0 ± 24.0   215 ± 14.0 *Positive control

In the formula 1 of compound, the R³ group is one of a hydrogen radicaland a glycosyl group, the R⁵ group is one of a hydrogen radical and ahydroxyl group, the R¹⁰ group is one selected from a group consisting ofa hydrogen radical, a hydroxyl group, a methyl radical, a formaldehydegroup and a formic acid radical, and the R¹⁶ group is one of a hydrogenradical and an acetic acid radical.

The formula 2 of compound, the R³ group is one of a hydrogen radical anda glycosyl group, the R¹⁰ group is one selected from a group consistingof a hydrogen radical, a hydroxyl group, a methyl radical, aformaldehyde group, and a formic acid radical.

R³ group is a special glycosyl group which are reevesiosyl group (a),4,6-dideoxy-2-O-methyl-β-D-allosyl group (b),6-deoxy-2-O-methyl-β-D-glycosyl group (c),6-deoxy-2-O-methyl-β-D-allosyl group (d).

The root of Reevesia formosana contains 13 cardenolide derivatives. Thecompounds, reevesiodides A-I, were assigned 1˜6,8,10 and 12, andepi-reevesiosides F-I were assigned 7, 9, 11 and 13. Structures of thereevesiosides A-F and the epi-reevesioside F belong to formula 1, andstructures of the reevesiosides G-I and the epi-reevesiosides G-I belongto formula 2.

substituted group Formula 1 R³ R⁵ R¹⁰ R¹⁶ reevesioside A (1) a OH CHO Hreevesioside B (2) a OH CHO OAc reevesioside C (3) b OH CHO Hreevesioside D (4) b OH COOH H reevesioside E (5) b OH OH H reevesiosideF (6) c H CH₃ H epi-reevesioside F (7) d H CH₃ H substituted groupFormula 2 R³ R¹⁰ reevesioside G (8) a (20S) CHO epi-reevesioside G (9) a(20R) CHO reevesioside H (10) a (20S) COOH epi-reevesioside H (11) a(20R) COOH reevesioside I (12) a (20S) OH epi-reevesioside I (13) a(20R) OH

According to the above mention that via bioassay-guided-fractionation,the EtOAc soluable fraction (Table. 1 and Table. 3) shows that the above13 ardenolide derivatives have cytotoxic effects on MCF-7, NCI-H460 andHepG2.

The term excipients or “pharmaceutically acceptable carrier orexcipients” and “bio-available carriers or excipients” mentioned aboveinclude any appropriate compounds known to be used for preparing thedosage form, such as the solvent, the dispersing agent, the coating, theanti-bacterial or anti-fungal agent and the preserving agent or thedelayed absorbent. Usually, such kind of carrier or excipient does nothave the therapeutic activity itself. Each formulation prepared bycombining the derivatives disclosed in the present invention and thepharmaceutically acceptable carriers or excipients will not cause theundesired effect, allergy or other inappropriate effects while beingadministered to an animal or human. Accordingly, the derivativesdisclosed in the present invention in combination with thepharmaceutically acceptable carrier or excipients are adaptable in theclinical usage and in the human. A therapeutic effect can be achieved byusing the dosage form in the present invention by the local orsublingual administration via the venous, oral, and inhalation routes orvia the nasal, rectal and vaginal routes. About 0.1 mg to 1000 mg perday of the active ingredient is administered for the patients of variousdiseases.

The carrier is varied with each formulation, and the sterile injectioncomposition can be dissolved or suspended in the non-toxic intravenousinjection diluents or solvent such as 1,3-butanediol. Among thesecarriers, the acceptable carrier may be mannitol or water. Besides, thefixing oil or the synthetic glycerol ester or di-glycerol ester is thecommonly used solvent. The fatty acid such as the oleic acid, the oliveoil or the castor oil and the glycerol ester derivatives thereof,especially the oxy-acetylated type, may serve as the oil for preparingthe injection and as the naturally pharmaceutical acceptable oil. Suchoil solution or suspension may include the long chain alcohol diluentsor the dispersing agent, the carboxylate methyl cellulose (CMC) or theanalogous dispersing agents, such as methyl cellulose, ethyl celluloseand hydroxylethyl methyl cellulose (HEMC). Other carriers are commonsurfactant such as Tween and Spans or other analogous emulsion, or thepharmaceutically acceptable solid, liquid or other bio-availableenhancing agent used for developing the formulation that is used in thepharmaceutical industry.

The composition for oral administration adopts any oral acceptableformulation, which includes capsule, tablet, pill, emulsion, aqueoussuspension, dispersing agent and solvent. The carrier is generally usedin the oral formulation. Taking the tablet as an example, the carriermay be the lactose, the corn starch and the lubricant, and the magnesiumstearate is the basic additive. The diluents used in the capsule includethe lactose and the dried corn starch. For preparing the aqueoussuspension or the emulsion formulation, the active ingredient issuspended or dissolved in an oil interface in combination with theemulsion or the suspending agent, and the appropriate amount of thesweetening agent, the flavors or the pigment is added as needed.

The nasal aerosol or inhalation composition may be prepared according tothe well-known preparation techniques. For example, the bioavailabilitycan be increased by dissolving the composition in the phosphate buffersaline and adding the benzyl alcohol or other appropriate preservative,or the absorption enhancing agent. The compound of the present inventionmay be formulated as suppositories for rectal or virginaladministration.

The compound of the present invention can also be administeredintravenously, as well as subcutaneously, parentally, muscular, or bythe intra-articular, intracranial, intra-articular fluid andintra-spinal injections, the aortic injection, the sterna injection, theintra-lesion injection or other appropriate administrations. Fordifferent patients, an administering dosage is between 0.1 mg to 0.2 mgof active components per day.

The above objectives and advantages of the present invention will becomemore readily apparent to those ordinarily skilled in the art afterreviewing the following detailed descriptions and accompanying drawings,in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a monocrystal x-ray diffractogram of the compound 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more specifically withreference to the following Embodiments. It is to be noted that thefollowing descriptions of preferred Embodiments of this invention arepresented herein for purpose of illustration and description only; it isnot intended to be exhaustive or to be limited to the precise formdisclosed.

Biological Experiments 1. Materials

MCF-7 (human breast adenocarcinoma), NCI-H460 (non-small-cell lungcancer) and HepG2 (liver hepatocellular cells) were incubated in amodified Dulbecco's Eagle's medium with 10% FBS and nonessential aminoacid (liver Technologies, Inc) in an incubator with 37° C., 5% CO₂/Air.Human cancer cell lines were planted in 96-well plates, and each wellwhich contained the 100 μl medium were planted one of MCF-7 and NCI-H460with a cell amount of 6500, 2500 and 7500. After overnight incubation,each cell line was administered at least 8 testing compounds withdifferent concentrations individually, and subsequently the cell lineswere incubated in the incubator for 72 hours. According to a method ofMTS reduction assay,5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoyl)-3-(4-sulfophenyl)tetrazolium salt (MTS) was applied on estimating alive cell numbers, and0.1% DMSO (Promega, Madison, Wis., USA) was a control group because DMSOcontrolled a result of percentage, the result could be a dose-responsecurve of IC₅₀ value. When the IC₅₀ value≦4 μg/ml, it was considered tohave an obvious cytotoxic effect. The IC₅₀ value was an average of threetimes repeats. The 10000/well HepG2 cells were planted on a 96-wellplate and were gone through the same experiences.

2. Extraction and Fractionation

6.5 kg dried roots of Reevesia formosana were cut into slices. Then, theslices were extracted by 30 L methanol for three times in the roomtemperature. After eliminating the solvent, the 150 g methanolicextractive was partitioned into an EtOAc fraction and a water fractionby EtOAc and H₂O, and 45 g EtOAc fraction and 100 g water fraction wereacquired. These two fractions were had active examination, and resultsshowed that the methanolic extractive had been shown cytotoxicityagainst MCF-7, NCI-H460 and HepG2 cancer cell lines.

The 45 g EtOAc fraction was subjected to a silicon column (70-230 sieve,1.5 kg), and a concentration gradient of n-hexane-EtOAc was applied onelution to acquire 1˜12 fractions, and at last methanol was used toelute to form 13 fractions totally. The fractions 9˜13 had been showncytotoxicity against MCF-7, NCI-H460 and HepG2 cancer cell lines.

The fraction 10 which was subjected to passage over a Sephadex LH-20column, eluting with 100% methanol, to yield 13 fractions (fractions10-1 to 10-13). The fraction 10-3 (0.87 g) was separated over a RP-C₁₈column (20-40 μm silicon), eluting with a mixing solvent of themethanol-water (3:2), and fractions 10-3-1 to 10-3-12 were obtained.Compound 1 (460 mg) was separated from the fraction 10-3-8, and acrystal mixture (10 mg) of compounds 8 and 9 was acquired from thefraction 10-3-5 by eluted with the solvent of n-hexane-EtOAc. Afterfiltering out the crystal, the solution was subjected to the siliconcolumn, eluting with the mixing solvent of CH₂Cl₂-acetone (5:1), toobtain 7 fractions (fractions 10-3-5-1 to 10-3-5-7).

The fraction 10-3-5-2 contained a mixture (1.7 mg) of compounds 12 and13, and the fraction 10-3-5-5 contained a mixture (7 mg) of compounds 10and 11. The fraction 10-3-6 (14.3 g) was subjected to the silicon column(15-35 μm silicon), eluting with a mixing solvent of CH₂Cl₂-acetone(3:1), to furnish 7 fractions (fractions 10-3-6-1 to 10-3-6-7). Thefraction 10-3-6-2 was purified by the RP-C₁₈ manufacturing TLC plate viathe mixing solvent of acetone-water (1:1), and then compound 2 (2 mg)was acquired. The fraction 10-3-10 was purified by the silicon column,eluting with a mixing solvent of CH₂Cl₂-acetone (3:1), to yield 5fractions (fractions 10-3-10-1 to 10-3-10-5). The fraction 10-3-10-3 andfraction 10-3-10-4 contained compounds 7 (63 mg) and 6 (11 mg),respectively. The fraction 11 (9 g) was applied to a Sephadex LH-20column and was eluted by 100% methanol, to afford 9 fractions (fractions11-1 to 11-9). The fraction 11-2 (0.74 g) was subjected to a RP-C₁₈column (20-40 μm silicon) using a mixing solvent of methanol-water (1:1)for elution, to obtain 14 fractions (fractions 11-2-1 to 11-2-14). Thefraction 11-2-8 (149 mg) was subjected to passage over a silicon column(15-35 μm silicon) eluting with a mixing solvent of CH₂Cl₂-acetone(3:1), to yield 9 fractions (fractions 11-2-8-1 to 11-2-8-9).

Besides, The fractions 11-2-8-7 and 11-2-8-8 were purified by themanufacturing TLC plate via the mixing solvent ofn-hexane-CH₂Cl₂-acetone (1:1:2), and then compounds 3 (9 mg), 4 (14 mg),5 (2.8 mg) and 6 (11 mg) were acquired.

3. Embodiment Preparation of Composition of Injection

According to quantities of the components, the components were measuredand taken to be dissolved in a liquid for preparation injections.

Reevesiosides A 0.135 mg/vial (1.1 mL)Liquid for infection qs

TABLE 4 Data of physical properties of the compounds 1~13 Reevesioside A(1): colorless prisms mp 231-232° C.; [α]²⁶ _(D) −28.7 (c 0.80, MeOH);IR (KBr) ν_(max) 3518 (OH), 1780, 1744, 1621 (butenolactone ring), 1715(CHO) cm⁻¹; ESIMS m/z 569 [M + Na]⁺; HRESIMS m/z 569.2729 (calcd forC₃₀H₄₂O₉Na, 569.2726). Reevesioside B (2): colorless syrup [α]²⁶ _(D)−18.7 (c 0.07, MeOH); IR (neat) ν_(max) 3511 (OH), 1775, 1741, 1628(butenolactone ring) cm⁻¹; ESIMS m/z 627 [M + Na]⁺; HRESIMS m/z 627.2777(calcd for C₃₂H₄₄O₁₁Na, 627.2781). Reevesioside C (3): colorless needlesmp 140-142° C.; [α]²⁶ _(D) −20.7 (c 0.08, MeOH); IR (KBr) ν_(max) 3498(OH), 1779, 1744, 1621 (butenolactone ring), 1712 (CHO) cm⁻¹; ESIMS m/z571 [M + Na]⁺; HRESIMS m/z 571.2881 (calcd for C₃₀H₄₄O₉Na, 571.2883).Reevesioside D (4): colorless needles mp 160-162° C.; [α]²⁶ _(D) −14.1(c 0.12, MeOH); IR (KBr) ν_(max) 3499 (OH), 1780, 1746, 1621(butenolactone ring), 2500-3300, 1730 (COOH) cm⁻¹; ESIMS m/z 587 [M +Na]⁺; HRESIMS m/z 587.2836 (calcd for C₃₀H₄₄O₁₀Na, 587.2832).Reevesioside E (5): colorless needles mp 228-229° C.; [α]²⁶ _(D) −10.3(c 0.04, MeOH); IR (KBr) ν_(max) 3479 (OH), 1779, 1745, 1620(butenolactone ring) cm⁻¹; ESIMS m/z 559 [M + Na]⁺; HRESIMS m/z 559.2886(calcd for C₂₉H₄₄O₉Na, 559.2883). Reevesioside F (6): colorless needlesmp 241-242° C.; [α]²⁶ _(D) −17.6 (c 0.08, MeOH); IR (KBr) ν_(max) 3442(OH), 1779, 1742, 1622 (butenolactone ring) cm⁻¹; ESIMS m/z 557 [M +Na]⁺; HRESIMS m/z 557.3088 (calcd for C₃₀H₄₆O₈Na, 557.3090).epi-Reevesioside F (7): colorless needles mp 216-217° C.; [α]²⁶ _(D)−16.9 (c 1.20; MeOH); IR (KBr) ν_(max) 3458 (OH), 1780, 1741, 1621(butenolactone ring) cm⁻¹; ESIMS m/z 557 [M + Na]⁺; HRESIMS m/z 557.3094(calcd for C₃₀H₄₆O₈Na, 557.3090). Mixed Reevesioside G (8) andepi-Reevesioside G (9): colorless syrup [α]²⁶ _(D) −46.7 (c 0.12, MeOH);IR (neat) ν_(max) 3524 (OH), 1780 (lactone ring), 1712 (CHO) cm⁻¹; ESIMSm/z 585 [M + Na]⁺; HRESIMS m/z 585.2672 (calcd for C₃₀H₄₂O₁₀Na,585.2676). Mixed Reevesioside H (10) and epi-Reevesioside H (11):colorless syrup [α]²⁶ _(D) −13.3 (c 0.20, MeOH); IR (neat) ν_(max) 3521(OH), 1780 (lactone ring), 2500-3300, 1727, (COOH) cm⁻¹; ESIMS m/z 601[M + Na]⁺; HRESIMS m/z 601.2621 (calcd for C₃₀H₄₂O₁₁Na, 601.2625). MixedReevesioside I (12) and epi-Reevesioside I (13): colorless syrup [α]²⁶_(D) −27.1 (c 0.10, MeOH); IR (neat) ν_(max) 3520 (OH), 1781 (lactonering) cm⁻¹; ESIMS m/z 573 [M + Na]⁺; HRESIMS m/z 573.2679 (calcd forC₂₉H₄₂O₁₀Na, 573.2676).

TABLE 5 ¹H NMR data of compounds 1~5^(a) position 1 2 3 4 5  1a/b 2.22,m/ 2.30, m/ 2.23, m/ 2.36, m/ 1.89, m/ 1.69, m 1.71, m 1.72, m 1.46, m1.49, m  2a/b 1.91, m/ 1.98, m/ 1.93, m/ 1.94, m/ 1.81, m/ 1.51, m 1.52,m 1.54, m 1.59, m 1.74, m  3 4.18, br s 4.21, br t 422, br s 4.25, br s4.17, br (2.7) t, (2.7)  4a/b 1.97, m/ 1.96, m/ 1.94, m/ 2.08, m/ 1.88,m/ 1.68, m 1.72, m 1.72, m 1.79, m 1.45, m  6a/b 2.06, m/ 2.04, m/ 2.07,m/ 1.77, m/ 1.86, m/ 1.68, m 1.71, m 1.72, m 1.69, m 1.45, m  7a/b 2.08,m/ 2.08, m/ 2.09, m/ 2.04, m/ 1.87, m/ 1.23, m 1.18, m 1.25, m 1.15, m1.06, m  8 1.91, m 1.97, m 1.92, m 1.98, m 1.81, m  9 1.51, m 1.43, m1.52, m 1.52, m 1.43, m 11a/b 1.50, m/ 1.51, m/ 1.54, m/ 2.26, m/ 1.58,m 1.30, m 1.38, m 1.31, m 1.86, m 12a/b 1.51, m/ 1.57, m/ 1.52, m/ 1.52,m/ 1.56, m/ 1.32, m 1.22, m 1.33, m 1.35, m 1.43, m 15a/b 2.00, m/ 2.63,dd 2.01, m/ 1.93, m/ 2.00, m/ 1.66, m (15.9, 9.6)/ 1.68, m 1.72, m 1.68,m 1.76, dd (15.9, 2.7) 16a/b 2.14, m/ 5.45, ddd 2.17, m/ 2.12, m/ 2.15,m/ 1.86, m (9.6, 8.6, 1.85, m 1.88, m 1.86, m 2.7) 17 2.75, dd 3.18, d(8.6) 2.75, dd 2.76, dd 2.77, dd (9.8, 5.4) (9.6, 5.2) (9.2, 4.8) (9.6,5.4) 18 0.85, s 0.94, s 0.86, s 0.97, s 0.92, s 19 10.0, s 10.0, d (0.8)10.05, s 21a/b 4.95, dd 4.95, dd 4.95, dd 4.98, dd 4.96, dd (18.0,(18.3, 1.8)/ (18.2, (18.2, 1.4)/ (18.0, 1.6)/ 4.85, dd 1.6)/ 4.81, dd1.5)/ 4.79, dd (18.3, 1.8) 4.79, dd (18.2, 1.4) 4.81, dd (18.0, (18.2,(18.0, 1.6) 1.6) 1.5) 22 5.86, s 6.00, s 5.88, s 5.88, s 5.88, s 251.98, s  1′ 4.46, d 4.47, d (6.9) 4.74, d 4.73, d 4.74, d (6.8) (7.9)(7.9) (8.0)  2′ 3.79, dd 3.82, dd 3.01, dd 3.03, dd 3.02, dd (6.8, 5.4)(6.9, 5.4) (7.9, 3.0) (7.9, 3.0) (8.0, 3.3)  3′ 4.13, 4.14, ddd 4.27, m4.30, m 4.28, m ddd (5.4, 4.2, (5.4, 4.0, 1.8) 1.8)  4′a/b 2.12, m/2.15, m/ 1.92, m/ 1.95, m/ 1.93, 1.76, m 1.72, m 1.49, m 1.49, m m/1.47,m  5′ 3.79, m 3.83, m 3.98, m 3.99, m 3.99, m  6′ 1.22, d 1.25, d (6.0)1.19, d 1.19, d 1.20, d (6.4) (6.0) (6.0) (6.6)  7′a/b 5.21, s/ 5.23, s/3.43, s 3.44, s 3.47, s 4.88, s 4.90, s OH-5^(b) 4.25, s 4.30, s 4.49, s4.39, s ^(a1)H NMR data (δ) were measured in CDCl₃ at 400 MHz for 1, 3,and 4, at 600 MHz for 2 and 5. ^(b)D₂O exchangeable

TABLE 6 ¹³C NMR data of compound 1~7^(a) position 1 2 3 4 5 6 7  1 17.818.0 17.9 21.3 28.6 30.3 30.2  2 25.3 25.3 25.4 25.7 26.1 26.6 26.6  373.0 73.1 72.0 72.2 72.4 73.2 73.1  4 34.5 34.4 34.0 33.2 34.3 29.5 29.5 5 73.4 73.2 73.1 74.6 73.71^(b) 36.4 36.3  6 36.3 36.3 36.5 36.5 34.526.6 26.6  7 24.1 23.7 24.2 23.9 23.5 21.4 21.3  8 41.6 41.5 41.7 40.740.5 41.8 41.7  9 39.3 39.1 39.3 39.4 39.8 35.8 35.7 10 54.6 54.3 54.653.3 73.74^(b) 35.2 35.1 11 21.9 21.4 21.9 21.8 21.0 21.1 21.1 12 39.739.0 39.7 40.0 40.0 40.0 39.9 13 49.4 49.8 49.4 49.8 49.5 49.6 49.6 1485.1 84.0 85.2 85.3 85.2 85.6 85.5 15 31.9 40.1 32.0 32.3 32.6 33.1 33.016 26.8 73.7 26.8 26.8 26.9 26.9 26.8 17 50.4 55.6 50.4 50.4 50.6 50.950.8 18 15.5 15.8 15.6 15.7 15.7 15.7 15.7 19 208.2 208.1 208.3 176.623.8 23.8 20 174.5^(b) 167.2 174.4^(b) 174.7^(b) 174.44^(c) 174.54^(b)174.8^(b) 21 73.3 75.5 73.4 73.6 73.4 73.4 73.5 22 117.7 121.6 117.9117.6 117.7 117.7 117.5 23 174.3^(b) 173.9 174.1^(b) 174.6^(b)174.38^(c) 174.53^(b) 174.7^(b) 24 170.5 25 21.1  1′ 99.1 99.3 96.5 96.596.5 100.6 97.8  2′ 73.7 73.8 80.7 80.5 80.7 83.3 80.2  3′ 74.5 74.664.6 64.3 64.6 76.3 69.9  4′ 34.3 34.3 38.1 38.1 38.1 75.2 72.8  5′ 67.067.1 66.5 66.7 66.5 71.4 69.5  6′ 20.9 20.9 20.6 20.6 20.6 17.6 17.7  7′95.4 95.4 57.6 57.3 57.6 60.8 59.4 ^(a13)C NMR data (δ) were measured inCDCl₃ at 100 MHz for 1, 3, 4, 6, and 7, at 150 MHz for 2 and 5.^(b, c)Interchangeable within the same column.

TABLE 7 ¹H NMR data of compounds 6~9^(a) Position 6 7 8 9  1a/b 1.50, m1.48, m 2.13, m/ 2.13, m/ 1.67, m 1.67, m  2a/b 1.69, m/ 1.66, m/ 1.92,m/ 1.92, m/ 1.52, m 1.48, m 1.48, m 1.48, m  3 4.08, br s 4.03, br s4.17, br s 4.17, br s  4a/b 1.70, m/ 1.69, m/ 1.95, m/ 1.95, m/ 1.23, m1.23, m 1.67, m 1.67, m  5 1.72, m 1.69, m  6a/b 1.88, m/ 1.84, m/ 2.18,m/ 2.18, m/ 1.28, m 1.26, m 1.67, m 1.67, m  7a/b 1.69, m/ 1.67, m/2.19, m/ 2.19, m/ 1.26, m 1.18, m 1.26, m 1.26, m  8 1.56, m 1.53, m1.90, m 1.90, m  9 1.61, m 1.59, m 1.38, m 1.38, m 11a/b 1.44, m/ 1.38,m/ 1.61, m/ 1.61, m/ 1.26, m 1.18, m 0.83, m 0.83, m 12a/b 1.52, m/1.51, m/ 1.67, m/ 1.67, m/ 1.39, m 1.40, m 1.42, m 1.42, m 15a/b 2.13,m/ 2.11, m/ 1.79, m/ 1.79, m/ 1.69, m 1.68, m 1.67, m 1.67, m 16a/b2.16, m/ 2.14, m/ 1.93, m/ 1.92, m/ 1.88, m 1.84, m/ 1.68, m 1.61, m 172.78, dd 2.76, dd 2.10, m 2.30, dd (8.8, 5.2) (9.2, 4.8) (9.2, 8.0)18a/b 0.87, s 0.85, s 4.13, d (10)/ 4.15, d (10)/ 3.40, d (10) 3.36, d(10) 19 0.93, s 0.91, s 10.05, s 10.05, s 21a/b 4.99, dd 4.98, dd 4.31,dd 4.34, s (18.1, 1.7)/ (18.0, 1.6)/ (10, 0.8)/ 4.81, dd 4.79, dd 3.97,d (10) (18.1, 1.7) (18.0, 1.6) 22a/b 5.88, s 5.85, s 2.64, d (17.4)/2.71, d (17.4)/ 2.57, dd 2.48, d (17.4) (17.4, 0.8)  1′ 4.34, d (7.7)4.64, d (7.8) 4.45, d (6.6) 4.45, d (6.6)  2′ 2.97, dd 3.02, dd 3.79, dd3.79, dd (6.6, (9.3, 7.7) (7.8, 3.0) (6.6, 5.6) 5.6)  3′ 3.42, dd 4.18,br t 4.12, m 4.12, m (9.3, 8.6) (3.0)  4′a/b 3.26, dd 3.23, ddd 2.11, m/2.11, m/ (9.3, 8.6) (9.6, 9.2, 1.71, m 1.71, m 3.0)  5′ 3.30, dq 3.61,dq 3.79, m 3.79, m (9.3, 6.2) (9.6, 6.4)  6′ 1.31, d (6.2) 1.25, d (6.4)1.21, d (6.0) 1.21, d (6.0)  7′a/b 3.62, s 3.54, s 5.20, s/ 5.20, s/4.87, s 4.87, s OH-3′^(b) 2.90, s OH-4′^(b) 2.62, br d (9.2) OH-5^(b)4.24, s 4.24, s OH-14^(b) 4.69, br s 4.69, br s ^(a) ¹H NMR data (δ)were measured in CDCl₃ at 400 MHz for 6-9. ^(b)D₂O exchangeable

TABLE 8 ¹³C NMR data of compounds 8~13^(a) position 8 9 10 11 12 13  117.8 17.8 21.8 21.8 28.6 28.6  2 25.4 25.4 25.6 25.6 26.1 26.1  3 72.972.9 73.4 73.4 73.3 73.3  4 34.5 34.5 33.9 33.9 34.3 34.3  5 73.1 73.174.6 74.6 73.6^(b) 73.6^(b)  6 36.2 36.2 36.2 36.2 34.7 34.7  7 24.524.5 24.3 24.3 23.8 23.8  8 43.7 43.8 42.1 42.1 42.0 42.1  9 39.5 39.439.62 39.57 39.69 39.66 10 54.6 54.6 53.3 53.3 73.9^(b) 73.9^(b) 11 24.524.5 23.6 23.6 23.5 23.5 12 36.3 36.4 36.5 36.7 36.6 36.7 13 58.7 58.859.0 58.8 58.8 58.9 14 83.6 83.6 83.8 83.8 83.7 83.7 15 34.3 34.2 35.034.8 35.0 34.8 16 25.3 23.7 25.2 23.6 25.4 23.5 17 54.9 57.0 54.9 57.055.1 57.2 18 71.3 71.5 71.5 71.7 71.6 71.8 19 207.9 207.9 176.0 176.0 2088.5 86.9 88.6 87.0 88.5 86.9 21 75.7 74.1 76.0 74.3 75.9 74.3 22 37.140.7 37.2 41.0 37.2 40.9 23 174.9 174.8 175.1 174.9 174.8 174.8  1′ 99.199.1 99.7 99.7 99.1 99.1  2′ 73.7 73.7 73.6 73.6 73.8 73.8  3′ 74.4 74.474.6 74.6 74.6 74.6  4′ 34.3 34.3 34.2 34.2 34.3 34.3  5′ 67.0 67.0 67.367.3 67.1 67.1  6′ 20.8 20.8 20.8 20.8 20.9 20.9  7′ 95.4 95.4 95.5 95.595.5 95.5 ^(a13)C NMR data (δ) were measured in CDCl₃ at 100 MHz for8-11, at 150 MHz for 12 and 13. ^(b)Interchangeable within the samecolumn.

TABLE 9 ¹H NMR Data of compounds 10-13^(a) position 10 11 12 13  1a/b2.28, m/1.82, m 2.28, m/1.82, m 1.93, m/1.47, m 1.93, m/1.47, m  2a/b1.92, m/1.51, m 1.92, m/1.51, m 1.87, m/1.76, m 1.87, m/1.76, m  3 4.22,br s 4.22, br s 4.15, br s 4.15, br s  4a/b 2.08, m/1.74, m 2.08,m/1.74, m 1.84, m/1.43, m 1.84, m/1.43, m  6a/b 1.89, m/1.62, m 1.89,m/1.62, m 1.90, m/1.43, m 1.90, m/1.43, m  7a/b 2.05, m/1.16, m 2.05,m/1.16, m 2.00, m/1.06, m 2.00, m/1.06, m  8 2.01, m 2.01, m 1.90, m1.90, m  9 1.43, m 1.43, m 1.31, m 1.31, m 11a/b 1.89, m/1.59, m 1.89,m/1.59, m 1.93, m/1.68, m 1.93, m/1.68, m 12a/b 1.74, m/1.43, m 1.74,m/1.43, m 1.81, m/1.53, m 1.81, m/1.53, m 15a/b 1.80, m/1.71, m 1.80,m/1.71, m 1.84, m/1.76, m 1.84, m/1.76, m 16a/b 1.89, m/1.67, m 1.77,m/1.64, m 1.87, m/1.76, m 1.90, m/1.67, m 17 2.08, m 2.28, m 2.15, m2.31, m 18a/b 4.26, d (10.4)/ 4.26, d (10.4)/ 4.23, d (10.2)/ 4.24, d(10.0)/ 3.51, d (10.4) 3.47, d (10.4) 3.48, d (10.2) 3.44, d (10.0)21a/b 4.33, d (10)/ 4.36, s 4.33, dd (10.2, 4.37, s 3.98, d (10) 0.8)/3.99, d (10.2) 22a/b 2.65, d (17.6)/ 2.74, d (17.2)/ 2.66, d (17.8)/2.74, d (17.2)/ 2.60, d (17.6) 2.44, d (17.2) 2.60, d (17.8) 2.45, d(17.2)  1′ 4.44, d (6.9) 4.44, d (6.9) 4.47, d (6.7) 4.47, d (6.7)  2′3.81, dd (6.9, 5.3) 3.81, dd (6.9, 5.3) 3.81, dd (6.7, 5.3) 3.81, dd(6.7, 5.3)  3′ 4.15, ddd 4.15, ddd 4.15, ddd 4.15, ddd (5.3, 3.6, 1.6)(5.3, 3.6, 1.6) (5.3, 3.6, 2.0) (5.3, 3.6, 2.0)  4′a/b 2.10, m/1.74, m2.10, m/1.74, m 2.14, m/1.76, m 2.14, m/1.76, m  5′ 3.80, m 3.80, m3.81, m 3.81, m  6′ 1.23, d (6.4) 1.23, d (6.4) 1.23, d (6.4) 1.23, d(6.4)  7′a/b 5.23, s/4.91, s 5.23, s/4.91, s 5.24, s/4.91, s 5.24,s/4.91, s ^(a) ¹H NMR data (δ) were measured in CDCl₃ at 400 MHz for 10and 11, at 600 MHz for 12 and 13.

There are more Embodiments provided as follows:

Embodiment 1: A pharmaceutical composition, includes:

an effective amount of a pharmaceutical compound being represented byformula (1):

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R⁵ group is one of a hydrogen radical and a hydroxyl group, the R¹⁰group is one selected from a group consisting of a hydrogen radical, ahydroxyl group, a methyl radical, a formaldehyde group and a formic acidradical, and the R¹⁶ group is one of a hydrogen radical and an aceticacid radical.

Embodiment 2: A pharmaceutical composition according to Embodiment 1includes an effective amount of at least one being selected from a groupconsisting of a reevesioside A, a reevesioside B, a reevesioside C, areevesioside D, a reevesioside E, a reevesioside F, and anepi-reevesioside F.

Embodiment 3: A pharmaceutical composition includes:

an effective amount of a pharmaceutical compound being represented byformula (2):

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R¹⁰ group is one selected from a group consisting of a hydrogenradical, a hydroxyl group, a methyl radical, a formaldehyde group, and aformic acid radical.

Embodiment 4: A pharmaceutical composition according to Embodiment 3includes an effective amount of at least one being selected from a groupconsisting of a reevesioside G, a epi-reevesioside G, a reevesioside H,a epi-reevesioside H, a reevesioside I and a epi-reevesioside I.

Embodiment 5: A pharmaceutical composition according to above Embodimentincludes a pharmaceutically acceptable carrier which is an excipientbeing one selected from a group consisting of a solvent, a dispersant, acoating, an antibacterial agent, an antifungal agent, a preservativeabsorbent, a delaying absorbent and a combination thereof.

Embodiment 6: A pharmaceutical composition according to above Embodimentis administered through one being selected from a group consisting of avein, an oral, an inspiration, a nasal cavity, a rectum, a vagina, ahypoglossis and a combination thereof.

Embodiment 7: A pharmaceutical composition according to above Embodimentis processed into one being selected from a group consisting of apowder, a capsule, a tablet, a pill and a combination thereof.

Embodiment 8: A pharmaceutical composition according to above Embodimentis extracted from a Reevesia formosana.

Embodiment 9: A pharmaceutical composition according to above Embodimentis used for treating a cancer.

Embodiment 10: A pharmaceutical composition according to aboveEmbodiment has a cytotoxic effect on cancer cells.

Embodiment 11: A cardenolide derivative, comprising a structure offormula 1

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R⁵ group is one of a hydrogen radical and a hydroxyl group, the R¹⁰group is one selected from a group consisting of a hydrogen radical, ahydroxyl group, a methyl radical, a formaldehyde group and a formic acidradical, and the R¹⁶ group is one of a hydrogen radical and an aceticacid radical.

Embodiment 12: The cardenolide derivatives according to Embodiment 11being selected from a group consisting of a reevesioside A, areevesioside B, a reevesioside C, a reevesioside D, a reevesioside E, areevesioside F and a epi-reevesioside F.

Embodiment 13: A cardenolide derivative, comprising a structure offormula 2:

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R¹⁰ group is one selected from a group consisting of a hydrogenradical, a hydroxyl group, a methyl radical, a formaldehyde group, and aformic acid radical.

Embodiment 14: The cardenolide derivatives according to Embodiment 13being extracted from a Reevesia formosana.

Embodiment 15: The cardenolide derivatives according to Embodiment 13further comprising an effective amount of at least one being selectedfrom a group consisting of a reevesioside G, an epi-reevesioside G, areevesioside H, an epi-reevesioside H, a reevesioside I and anepi-reevesioside I.

Embodiment 16: The cardenolide derivatives as claimed in claim 15 usedfor treating a cancer.

1. A pharmaceutical composition, comprising: an effective amount of apharmaceutical compound being represented by formula 1:

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R⁵ group is one of a hydrogen radical and a hydroxyl group, the R¹⁰group is one selected from a group consisting of a hydrogen radical, ahydroxyl group, a methyl radical, a formaldehyde group and a formic acidradical, and the R¹⁶ group is one of a hydrogen radical and an aceticacid radical.
 2. The pharmaceutical composition as claimed in claim 1further comprising an effective amount of at least one being selectedfrom a group consisting of a reevesioside A, a reevesioside B, areevesioside C, a reevesioside D, a reevesioside E, a reevesioside F andan epi-reevesioside F.
 3. The pharmaceutical composition as claimed inclaim 1, wherein the pharmaceutically acceptable carrier is an excipientbeing one selected from a group consisting of a solvent, a dispersant, acoating, an antibacterial agent, an antifungal agent, a preservativeabsorbent, a delaying absorbent and a combination thereof.
 4. Thepharmaceutical composition as claimed in claim 1 being administeredthrough one being selected from a group consisting of a vein, an oral,an inspiration, a nasal cavity, a rectum, a vagina, a hypoglossis and acombination thereof.
 5. The pharmaceutical composition as claimed inclaim 1 being processed into one being selected from a group consistingof a powder, a capsule, a tablet, a pill and a combination thereof. 6.The pharmaceutical composition as claimed in claim 1 being extractedfrom a Reevesia formosana.
 7. The pharmaceutical composition as claimedin claim 1 used for treating a cancer.
 8. The pharmaceutical compositionas claimed in claim 1 having a cytotoxic effect on cancer cells.
 9. Apharmaceutical composition, comprising: an effective amount of apharmaceutical compound being represented by formula 2:

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R¹⁰ group is one selected from a group consisting of a hydrogenradical, a hydroxyl group, a methyl radical, a formaldehyde group, and aformic acid radical.
 10. The pharmaceutical composition as claimed inclaim 9 further comprising an effective amount of at least one beingselected from a group consisting of a reevesioside G, anepi-reevesioside G, a reevesioside H, an epi-reevesioside H, areevesioside I and an epi-reevesioside I.
 11. The pharmaceuticalcomposition as claimed in claim 9, wherein the pharmaceuticallyacceptable carrier is an excipient being one selected from a groupconsisting of a solvent, a dispersant, a coating, an antibacterialagent, an antifungal agent, a preservative absorbent, a delayingabsorbent and a combination thereof.
 12. The pharmaceutical compositionas claimed in claim 9 being extracted from a Reevesia formosana.
 13. Thepharmaceutical composition as claimed in claim 9 used for treating acancer.
 14. The pharmaceutical composition as claimed in claim 9 havinga cytotoxic effect on cancer cells.
 15. A cardenolide derivative,comprising a structure of formula 1:

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R⁵ group is one of a hydrogen radical and a hydroxyl group, the R¹⁰group is one selected from a group consisting of a hydrogen radical, ahydroxyl group, a methyl radical, a formaldehyde group and a formic acidradical, and the R¹⁶ group is one of a hydrogen radical and an aceticacid radical.
 16. The cardenolide derivatives as claimed in claim 15being selected from a group consisting of a reevesioside A, areevesioside B, a reevesioside C, a reevesioside D, a reevesioside E, areevesioside F and an epi-reevesioside F.
 17. The cardenolidederivatives as claimed in claim 15 being extracted from a Reevesiaformosana.
 18. The cardenolide derivatives as claimed in claim 15 usedfor treating a cancer.
 19. A cardenolide derivative, comprising astructure of formula 2:

wherein the R³ group is one of a hydrogen radical and a glycosyl group,the R¹⁰ group is one selected from a group consisting of a hydrogenradical, a hydroxyl group, a methyl radical, a formaldehyde group, and aformic acid radical.
 20. The cardenolide derivatives as claimed in claim19 further comprising an effective amount of at least one being selectedfrom a group consisting of a reevesioside G, an epi-reevesioside G, areevesioside H, an epi-reevesioside H, a reevesioside I and anepi-reevesioside I.
 21. The cardenolide derivatives as claimed in claim19 being extracted from a Reevesia formosana.
 22. The cardenolidederivatives as claimed in claim 19 used for treating a cancer.